Saccharifying cellulose

ABSTRACT

Dissolution, partial dissolution or softening of cellulose in an ionic liquid (IL) and its subsequent contact with anti-solvent produces regenerated cellulose more amorphous in structure than native cellulose, which can be separated from the IL/anti-solvent mixture by mechanical means such as simple filtration or centrifugation. This altered morphology of IL-treated cellulose allows a greater number of sites for enzyme adsorption with a subsequent enhancement of its saccharification. The IL-treated cellulose exhibits significantly improved hydrolysis kinetics with optically transparent solutions formed after about two hours of reaction. This provides an opportunity for separation of products from the catalyst (enzyme) easing enzyme recovery. With an appropriate selection of enzymes, initial hydrolysis rates for IL-treated cellulose were up to two orders of magnitude greater than those of untreated cellulose. Due to the non-volatility of the IL, anti-solvent can be easily stripped from the IL/anti-solvent mixture for recovery and recycle of both the ionic liquid and anti-solvent.

TECHNICAL FIELD

This invention relates to saccharifying cellulose from biomass.Cellulose is the most abundant renewable resource in the world. It is amajor fraction of plant biomass, which is the feedstock, for “futurebiorefineries” with the potential to replace the conventionalpetrochemical refineries in an economy based on renewable resources.

BACKGROUND OF THE INVENTION

The utilization of lignocellulosic waste materials, such as cornstalks,sawdusts, straws, bagasse, and the like, has been the subject of stronginterest recently, particularly with respect to utilization of suchagricultural and waste materials for developing alternate sources offuels, chemicals, glucose and the like. Lignocellulose is commonlyreferred to as biomass.

Lignocellulosic materials include three principal components—cellulose(30-40%), hemicellulose (20-30%), and lignin (5-30%). In its naturalstate, cellulose is highly crystalline in structure with individualcellulose polymer chains held together by strong hydrogen bonding andvan der Waals forces. The individual cellulose chains are linearcondensation polymer molecules made up of anhydroglucose units joinedtogether by β-1,4 glycosidic bonds with degrees of polymerization (DP)ranging, typically, from 1,000 to 15,000 units. The high crystallinityof cellulose, while imparting structural integrity and mechanicalstrength to the material, renders it recalcitrant towards hydrolysisaimed at producing glucose—the feedstock for producing fuels andchemicals—from this polysaccharide. In general, neither the watermolecules nor the catalysts for hydrolysis (saccharification) are ableto easily penetrate the crystalline matrix.

Cellulose hydrolysis to glucose is most often catalyzed using mineralacids or enzymes (cellulases). Cellulase hydrolysis is preferred overmineral acid hydrolysis for several reasons: acid hydrolysis leads toformation of undesirable degradation products of glucose thatsignificantly lower glucose yield and inhibit subsequent fermentation;requires expensive corrosion-resistant materials; and poses disposalproblems. Glucose degradation products observed with acid pretreatmentor hydrolysis include hydroxymethyl furfural (HMF) and furfural whichinhibit downstream fermentation to ethanol.

On the other hand, cellulase enzymes are very specific in their action,producing virtually no glucose degradation products. Cellulases (offungal or bacterial origin) are in fact a mixture of enzymes which actin concert and synergistically. Special materials of construction arenot required with cellulase-catalyzed hydrolysis. However, cellulosehydrolysis in aqueous media suffers from slow reaction rates because thesubstrate (cellulose) is a water-insoluble crystalline biopolymer.Therefore, the enzymes have to accomplish the hydrolytic decompositionvia first adsorbing on the cellulose surface, partially stripping theindividual polymer chains from the crystal structure, and then cleavingthe glycoside bonds in the chain. Adsorption sites of crystallinecellulose are very limited due to the tight packing arrangement ofcellulose fibrils which not only excludes the enzymes but also largelyexcludes water.

Cellulose is very difficult to dissolve due to the extensive network ofinter and intra-molecular bonds and interactions between cellulosefibrils. Ionic liquids have recently been shown to be novel solvents fordissolution of cellulose capable of dissolving large amounts ofcellulose at mild conditions (Swatloski, R. P. et al., J Am. Chem. Soc.,2002, 124, 4974-4975; Zhang, H. et al., Macromolecules, 2005, 38,8272-8277). Ionic liquids (ILs) are salts that typically melt below˜100° C. With their low volatility, fluidity at ambient temperatures,and unique solvent properties, ILs comprise a class of prospectivesolvents that are potentially ‘green’ due to their minimal airemissions. Our invention exploits these properties of ionic liquids toenhance the saccharification of cellulose.

SUMMARY OF THE INVENTION

We have now invented an efficient route for saccharifying cellulose frombiomass for fuel and chemical production.

Hydrolysis of cellulose to glucose is critically important in producingfuels and chemicals from renewable feedstocks. Cellulose hydrolysis inaqueous media suffers from slow reaction rates because cellulose is awater-insoluble crystalline biopolymer. To accomplish its hydrolysis,the hydrolyzing enzymes (cellulases) and water must penetrate thecrystalline fibrils. Pretreatment methods which increase the surfacearea accessible to water and cellulases are vital to improving thehydrolysis kinetics and conversion of cellulose to glucose. In a noveltechnique, the cellulose is dissolved or partially dissolved in an ionicliquid (IL) and subsequently recovered as an amorphous precipitate or asolid of lower crystallinity index by rapidly quenching the solutionwith an anti-solvent. Hydrolysis kinetics of the recovered cellulose aresignificantly enhanced. Because of their extremely low volatility, ionicliquids are expected to have minimal environmental impact. With anappropriate selection of enzymes, initial hydrolysis rates for recoveredcellulose were up to ninety times greater than those of untreatedcellulose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: is a schematic diagram of a unit for the regeneration ofcellulose with ionic liquids.

FIG. 2: shows X-ray powder diffraction patterns of untreated andregenerated Avicel. Untreated Avicel, (A), exhibited a significantlygreater degree of crystallinity than that of regenerated samples (B)through (D). Regenerated samples were incubated in BMIMCI at 130° C. fortwo hours and precipitated with (B) deionized water, (C) methanol, or(D) ethanol.

FIGS. (3 a) and (3 b) show hydrolysis rates of IL incubated samplescompared to those of untreated Avicel (

). 5% (□), 10% (∘), 15% (Δ), 30% (∇) Avicel samples were incubated for30 minutes in AMIMCI at 120° C., and precipitated with deionized water.Conversion of cellulose to sugars for batch samples of ˜17 mg/ml Avicelhydrolyzed with T. reesei cellulase activity of 16 FPU/g glucan andsupplemental cellobiase activity of 83 CBU/g glucan at 50° C. is shownas a function of time for (3 a) total soluble sugars (measured using aDNS assay) and (3 b) as % cellulose conversion to glucose (measured byHPLC).

FIGS. (4 a) and (4 b) show hydrolysis rates of IL incubated samplescompared to those of untreated Avicel (

). 5% (□), 10% (∘), 15% (Δ), 30% (∇) Avicel samples were incubated for30 minutes in BMIMCI at 120° C., and precipitated with deionized water.Conversion of cellulose to sugars for batch samples of 17 mg/ml Avicelhydrolyzed with T. reesei cellulase activity of 16 FPU/g glucan andsupplemental cellobiase activity of 83 CBU/g glucan at 50° C. is shownas a function of time for (4 a) total soluble sugars (measured using aDNS assay) and (4 b) as % cellulose conversion to glucose (measured byHPLC).

DETAILED DESCRIPTION OF THE INVENTION

The goal of this pretreatment invention is to open the structure ofcellulose to make it accessible to water and the component enzymes ofcellulases in order to increase the rate of hydrolysis of cellulose toglucose or soluble glucose oligomers. In our approach, ionic liquids areused to dissolve or partially dissolve crystalline cellulose. Any ionicliquid that is capable of dissolving cellulose can be used in thepretreatment strategy outlined here. The disclosure of U.S. Pat. No.6,824,599 herein is incorporated by reference. The cellulose dissolutionstep is followed by rapid quenching with an “anti-solvent” such aswater, acetonitrile or alcohol, the high affinity of which towards theIL forces the latter to reject all the dissolved cellulose via apreferential solute-displacement mechanism. The resulting cellulose iseither an amorphous precipitate or a solid of significantly lowercrystallinity depending on whether the initial cellulose weight percentis lower or higher than its solubility limit in the IL. The rejectedcellulose can be separated from the IL-anti-solvent solution throughmechanical separations such as centrifugation or filtration. The ionicliquids are nonvolatile and they can be recovered from theanti-solvent/IL mixture by flash distillation, solvent extraction,membranes or ion exchange techniques (FIG. 1).

The recovered cellulose is referred to as regenerated cellulose (RC)when it is an amorphous precipitate, as is the case when the cellulosecompletely dissolves in the IL during incubation. However, when theinitial cellulose weight percent is more than its solubility limit inthe IL, only partial dissolution of cellulose occurs during incubation.Subsequent anti-solvent treatment provides a cellulose mix of RC andIL-swollen partially crystalline cellulose (PCC). In what follows,cellulose samples recovered following anti-solvent treatment arereferred to as “IL-treated cellulose” irrespective of whether theresulting cellulose is RC or a mixture of RC and PCC. In solvent-swollencellulose the degree of crystallinity of cellulose is progressivelyreduced as the extent of swelling increases but is not eliminated,whereas regenerated-cellulose is essentially amorphous. The hydrolysisrates of cellulose depend on the extent of swelling with the maximalimprovement in hydrolysis with amorphous regenerated-cellulose.

The amount of cellulose that can be completely dissolved in the ILduring incubation was shown to vary depending on whether the mixture wassubjected to conventional heating with agitation or microwaveirradiation with intermittent agitation. The latter procedure was shownto be capable of bringing larger amounts of cellulose to completedissolution compared to conventional heating (Swatloski, R. P. et al.,J. Am. Chem. Soc., 2002, 124, 497-44975). Regardless of which approachis taken for dissolving cellulose, quenching the solution with ananti-solvent for cellulose renders the precipitate essentiallyamorphous, providing significant improvement in hydrolysis. Thus, theproposed invention of using IL-pretreatment to obtain readilyhydrolysable cellulose can be benefited by all means capable ofdissolving large amount of cellulose in the IL.

EXAMPLE 1 Structure of Regenerated Cellulose (RC)

X-ray powder diffraction (XRD) data were obtained for IL-treatedcellulose to assess crystallinity of recovered samples. Cellulose wasfully dissolved in BMIMCI before precipitation with addition ofanti-solvent for experiments outlined in this example. Anti-solventselection, incubation time and temperature were varied.

Micro-crystalline cellulose, Avicel PH-101 (FMC Corp., Philadelphia,Pa.) was obtained from Sigma Aldrich, St. Louis, Mo. A 5% (w/w)cellulose solution was prepared by combining 50 mg of cellulose with 950mg of 1-n-butyl-3-methylimidazolium chloride BMIMCI in a 5 ml autoclavevial. The vial and the contents were heated in a block heater to 130° C.and incubated for 10 minutes. The samples were gently stirred by placingthe block heater on an orbital shaker.

Water, methanol and ethanol were used as anti-solvents for precipitatingcellulose from BMIMCI. About 2 ml of anti-solvent was added to thecellulose/BMIMCI mixture. A precipitate immediately formed. The samplewas briefly centrifuged and supernatant was removed. The sample waswashed five to six times with additions of anti-solvent, centrifuged andsupernatant removed. Smooth films for X-ray powder diffraction (XRD)data collection were cast at room temperature with RC and untreatedcellulose on microscope slides. XRD data for these films were generatedat 25° C. with an XPERT' PRO powder diffractometer with an Xcelerator'detector (PANalytical, ALMELO, The Netherlands) using Nickel filteredCuKα radiation. Samples were scanned over the angular range 6.0-45.0°,2θ, with a step size of 0.05°, and step time of 10 seconds.

As seen in FIG. 2 cellulose regenerated with all the selectedanti-solvents produced amorphous cellulose whereas the untreatedcellulose is highly crystalline. In additional XRD experiments,incubation time and temperature were varied from 10 to 180 minutes andfrom 130 to 150° C. with complete dissolution of cellulose (Dadi, A. P.,S. Varanasi, C. A. Schall (2006) Biotechnology and Bioengineering,95(5), 904-910). The resulting regenerated cellulose structure wasamorphous. XRD results suggest that during pretreatment with IL andanti-solvent cellulose crystallinity is disrupted. Rapid precipitationwith anti-solvent may prevent the restructuring of the dissolvedcellulose into its crystalline form.

EXAMPLE 2 Initial Hydrolysis Rates of Regenerated Cellulose by Cellulase

For hydrolysis experiments, Avicel was regenerated by dissolution inBMIMCI followed by precipitation with water, methanol, or ethanol.Alternative anti-solvents methanol and ethanol were examined becauseBMIMCI is more easily recovered, following cellulose regeneration, fromvolatile organic solvents than water through a simple distillation step.Batch enzymatic hydrolysis of regenerated and untreated cellulose wascarried out at 50° C. in a reciprocating shaker bath. Total batch volumewas approximately 3 ml with a cellulase enzyme concentration of 60 FPU/gglucan, and substrate concentration of about 17 mg/ml. Solutions werebuffered with 0.05M sodium citrate, pH 4.8. The enzyme reaction wasmonitored by withdrawing samples from the supernatant periodically andmeasuring release of soluble reducing sugars by the DNS assay (Miller GL. (1959) Anal. Chem. 31(3):426-428). Untreated and regeneratedcellulose were hydrolyzed using the same cellulase (from T. reesei)stock solution. The untreated cellulose controls were run concurrentlywith all regenerated cellulose hydrolysis experiments to eliminatepotential differences in temperature history or enzyme loading.

The resulting initial rates of hydrolysis of regenerated and untreatedcellulose to soluble reducing sugars as measured by total solublereducing sugars are shown in Table 1. Initial rates of enzymatichydrolysis of regenerated cellulose were at least fifty times that ofuntreated cellulose. All anti-solvents studied appear to lead to similarenhancement in initial saccharification rates (Table 1).

TABLE 1 Initial rate of formation of total soluble reducing sugarsmeasured by DNS assay in enzymatic hydrolysis of Avicel cellulose.Initial rate of formation of soluble reducing sugars Rate Anti-solvent(mg ml⁻¹min⁻¹) Enhancement* water 0.6473 52 methanol 0.6823 55 ethanol0.6473 53 untreated 0.0125 — 17 mg/ml of regenerated or untreated Avicelsamples were hydrolyzed using a cellulase activity of 60 FPU/g glucan.Regenerated cellulose samples were incubated in BMIMCI for 10 minutes at130° C. and precipitated using the anti solvents water, methanol, orethanol. Rates are calculated from data obtained in the first 20 minutesof hydrolysis. (Dadi, A. P., S. Varanasi, C. A. Schall (2006)Biotechnology and Bioengineering, 95(5), 904-910) *Rate enhancement isdefined as the ratio of initial rate of reducing sugars released forregenerated cellulose divided by that of untreated cellulose.

EXAMPLE 3 Initial Hydrolysis Rates of Regenerated Cellulose byCellulases in the Presence of Additional β-Glucosidase

In this example the effect of augmenting cellulase (Celluclast 1.5L, aTrichoderma reesei cellulase) with additional β-glucosidase (Novozyme188, a cellobiase) on the rates of hydrolysis of untreated andregenerated cellulose was investigated. The enzymes were obtained fromNovozyme Corp., Bagsvaerd, Denmark. Cellulase activity was determined bythe standard filter paper assay and expressed as filter paper units(FPU) per gram of glucan (Gosh, T. K., (1987), Pure Appl. Chem., 59,257-268). Cellobiase activity was determined by a cellobiose hydrolysisassay (Gosh, T. K., (1987), Pure Appl. Chem., 59, 257-268) and expressedas cellobiose units (CBU) per gram of glucan.

A batch volume of 3 ml with a cellulose concentration of 16.7 mg/ml wasused with both untreated cellulose and regenerated-cellulose (recoveredfrom 5% (w/w) cellulose-BMIMCI mixture using de-ionized water asanti-solvent). The enzyme loadings were varied from 8 to 32 FPU/g glucanof Celluclast 1.5L and 0 to 83 CBU/g glucan of Novozyme 188. The enzymereaction was monitored by withdrawing 20 μl samples from the supernatantperiodically. Withdrawn samples were diluted 10 times and heated to 100°C. for 5 minutes. Untreated and IL-treated cellulose were hydrolyzedusing the same cellulase and β-glucosidase stock solutions. Theuntreated-cellulose controls were run concurrently with all theIL-treated-cellulose hydrolysis experiments to eliminate potentialdifferences in temperature history or enzyme loading.

The released reducing sugars were measured by the dinitrosalicylic acid(DNS) method using D-glucose as a standard. Released glucose wasdetermined separately by high performance liquid chromatography (HPLC)using a HPX-87 P column (Bio-Rad Laboratories Inc., Hercules, Calif.) at80° C. equipped with a refractive index detector. The mobile phase wasdeionized water with a flow rate of 0.6 ml/minute.

The initial rate of soluble reducing sugar formation of untreatedcellulose and cellulose regenerated from a 5% cellulose/BMIMCI mixtureis shown in Table 2 for various enzyme loadings, with and withoutβ-glucosidase addition.

TABLE 2 Effect of additional β-glucosidase on hydrolysis. Initial rateof formation of total reducing sugars, measured by DNS assay. Enzymeactivity per g of glucan Initial rate (mg ml⁻¹min⁻¹) Cellulaseβ-glucosidase Untreated Regenerated Rate (FPU) (CBU) Cellulose CelluloseEnhancement* 8 0 0.0004 0.0047 12 8 83 0.0004 0.0320 71 16 0 0.00430.0427 10 16 83 0.0044 0.3915 89 32 0 0.0110 0.3953 36 32 83 0.01400.5030 36 Rates are calculated from analysis of supernatant sampledduring the first 20 minutes of hydrolysis. Regenerated cellulose wasformed by incubating samples of 5% cellulose in ionic liquids (BMIMCI)at 130° C. for 10 minutes followed by precipitation with water. *Rateenhancement is defined as the ratio of initial rate of reducing sugarsreleased for regenerated cellulose divided by that of untreatedcellulose.

The rate enhancement, defined as the ratio of initial hydrolysis rate ofIL-treated cellulose to that of untreated cellulose, appears highest foran enzyme loading of 16 FPU/g glucan with addition of β-glucosidase at83 CBU/g glucan. At these enzyme loadings the hydrolysis rate ofregenerated cellulose is nearly two orders of magnitude greater thanthat of untreated cellulose. For modest cellulase activities (8 and 16FPU/g glucan), the hydrolysis rates of regenerated cellulose increasedsignificantly with addition of β-glucosidase (by 6 to 9 fold). Thisincrease was not seen in untreated cellulose samples at similarcellulase activities (Table 2).

EXAMPLE 4 Effect of the Residual Crystallinity of IL-Treated Celluloseon Hydrolysis

In this example, Avicel and BMIMCI or 1-allyl-3-methyl imidazoliumchloride (AMIMCI) mixtures containing 5%, 10%, 15% and 30% (w/w)cellulose were incubated in a 5 ml autoclave vial. The vial and thecontents were heated in a block heater to 120 to 130° C. for 10 to 30minutes. The samples were gently stirred by placing the block heater onan orbital shaker.

Deionized water was used as an anti-solvent for recovering cellulosefrom the ionic liquids, BMIMCI and AMIMCI. About 2 ml of anti-solventwas added to the cellulose/ionic liquid mixture. A precipitateimmediately formed. The sample was briefly centrifuged and supernatantwas removed. The precipitated sample was washed with additional aliquotsof water followed by the cellulose hydrolysis buffer solution.

During the incubation of Avicel in ionic liquids at 120° C. and 130° C.,complete dissolution was observed for 5% (w/w) Avicel solutions inBMIMCI and AMIMCI. 10% solutions were completely dissolved in BMIMCI andalmost completely dissolved in AMIMCI. For 15 and 30% (w/w) Avicel inILs, only partial dissolution occurred. The maximum solubility of Avicelobserved visually at 120° C. was 9% in AMIMCI and 13% in BMIMCI.

Samples of cellulose recovered following anti-solvent treatment ofIL-cellulose mixtures and untreated-cellulose were examined by XRD.Crystallinity index, CrI, was determined from X-ray powder diffractiondata (Segal, L., Creely, J. J., Martin, A. E., and Conrad, C. M. (1959)Text. Res. J. 29) and calculated using the formula:CrI=[(I₀₂₀−I_(am))/I_(020]×100) where I₀₂₀ is the intensity abovebaseline at the 020 peak maximum near 2θ 22.5° and I_(am) is the minimumin peak intensity near 2θ of 18°. A reduction in CrI was observed forall samples incubated in the ILs (Table 3). For cellulose concentrationsbelow the solubility limit in the IL (5 and 10% (w/w)), the celluloserecovered following anti-solvent addition is essentially amorphous.Accordingly, the reduction in the measured CrI was greatest for 5 and10% (w/w) samples and remains essentially the same for both ILs.However, with samples containing initial cellulose weight percent aboveits solubility limit in the IL (15 and 30% (w/w)), only partialdissolution of cellulose occurs during incubation. Subsequentanti-solvent treatment provides a cellulose mix of regenerated cellulose(RC) and partially crystalline cellulose (PCC). The proportion of PCC isexpected to rise as the initial wt % of cellulose incubated in IL isincreased. This gradual increase in PCC will lead to a correspondingincrease in CrI as was observed from the CrI obtained from XRDmeasurement (Table 3).

TABLE 3 Effect of Crl of IL-treated Avicel on hydrolysis. Initial rateof formation of total soluble reducing sugars, measured by DNS assayduring the enzymatic hydrolysis of Approx 17 mg/ml Avicel (with acellulase activity of 16 FPU/g glucan and added β-glucosidase activityof 83 CBU/g glucan). Crystallinity Concentration Initial rate Rate Indexof Avicel in IL (mg ml⁻¹min⁻¹) Enhancement* (Crl) Untreated 0.0046 —76.4  5% in AMIMCI 0.3274 71 12.9 10% in AMIMCI 0.3397 74 11.7 15%AMIMCI 0.2304 50 15 30% AMIMCI 0.1263 27 47.0  5% in BMIMCI 0.3412 7411.5 10% BMIMCI 0.3763 82 11.6 15% BMIMCI 0.289 63 14.2 30% BMIMCI0.2140 46 43.4 Rates are calculated from analysis of supernatant sampledduring the first 20 minutes of hydrolysis. RC or a mixture of RC & PCCwas formed by incubating cellulose in ionic liquids (AMIMCI/BMIMCI) at120° C. for 30 minutes followed by contact with water. A mixture of RC &PCC formed at Avicel concentrations in IL above 10% (w/w). *Rateenhancement is defined as the ratio of initial rate of reducing sugarsreleased for regenerated cellulose divided by that of untreatedcellulose.

The effect of crystallinity of recovered cellulose on hydrolysis wasalso investigated in this example. Batch volumes were adjusted forIL-treated cellulose samples to achieve the same cellulose concentrationof 16.7 mg/ml employed with untreated cellulose. The resulting volumeswere 3 ml, 6 ml, 9 ml and 18 ml, respectively, for cellulose samplesrecovered from IL-cellulose mixtures of 5%, 10%, 15% and 30% (w/w)cellulose. A constant enzyme loading of 16 FPU/g glucan with addition ofβ-glucosidase at 83 CBU/g glucan was used in all experiments conductedwith various IL-treated-cellulose samples (incubation of 5, 10, 15 and30% cellulose in the ionic liquids). The initial rates of hydrolysis foruntreated and IL-treated-cellulose are shown in Table 3. Theconcentrations of total soluble sugars and glucose are shown asfunctions of time in FIGS. 3 and 4.

Initial rates of enzymatic hydrolysis of completely dissolved andregenerated cellulose samples (5 and 10%) were higher than theIL-treated-cellulose samples that were partially dissolved (15 and 30%)as seen in Table 3. Samples containing initial cellulose concentrationsbelow 10% are within the solubility limit in IL, and above 10% are abovethe solubility limit in IL. Addition of anti-solvent to IL-cellulosemixtures produced regenerated-cellulose (RC) when the celluloseconcentration is within the solubility limit and produced a mixture ofRC and partially crystalline cellulose (PCC) above the solubility limit.Mixtures of RC and PCC have residual crystallinity (Table 3) whichaccounted for lower initial rates compared to RC samples.

For both RC (5% & 10%) and a mixture of RC and PCC (15% & 30%) samples,conversion to glucose after 7 hours of hydrolysis was about 80-85%whereas it is only 20% for untreated cellulose (FIGS. 3 b and 4 b).IL-treated-cellulose exhibited improved hydrolysis kinetics withoptically transparent solutions formed after first few hours of reaction(within ˜2 to 4 hours), indicating relatively fast hydrolysis kineticsand rapid conversion of cellulose into soluble oligomers of glucose andcellobiose. In contrast with untreated cellulose, solid cellulose ispresent even after extended hydrolysis times (greater than 24 hours).Cellulase and other enzymes are adsorbed on to solid cellulosesubstrate, resulting in difficult recovery and loss of these valuableenzymes. With the absence of residual solid cellulose in hydrolysatesproduced with IL-treated cellulose, enzymes are easily recovered.

Higher conversions were expected for RC samples as the crystallinity ofcellulose is almost eliminated in RC samples. Higher conversionsobtained for mixtures of RC and PCC (15% & 30%) are somewhat surprising.In spite of the residual crystallinity of these cases, the conversionswere higher and comparable to that of RC samples (5% & 10%) (FIGS. 3 &4). This implies that the crystallinity of 15% and 30% cellulose samplestreated in IL was reduced sufficiently to provide enough accessiblesites for cellulase enzyme adsorption and activity. This is a promisingobservation as it suggests that it is not necessary to “totallyeliminate” the crystallinity of cellulose to achieve significantenhancement in hydrolysis rates, and even with appreciable residualcrystallinity most of the recalcitrance to hydrolysis can be mitigated.This also offers the possibility to process larger amounts of celluloserapidly (i.e., up to 30 wt % of cellulose can be incubated in theIL-treatment step). All IL-treated cellulose samples reached almost 95%conversions to glucose within 24 h whereas untreated cellulose onlyreached 50% conversion in that time period (FIGS. 3 & 4).

The hydrolysis rates of IL-treated-cellulose samples within thesolubility limit (5% & 10%) were comparable for cellulose incubated ineither AMIMCI or BMIMCI. With IL-treated-cellulose samples above thesolubility limit (15% & 30%), the hydrolysis rates appear to differaccording to the dissolving capabilities of the ionic liquid. Thehydrolysis rates of IL-treated-cellulose prepared from 15% and 30%Avicel incubation in BMIMCI were higher than those prepared with AMIMCI(Table 3).

SUMMARY & CONCLUSIONS

In a novel technique, the microcrystalline cellulose was incubated withan ionic liquid (IL) and then recovered as essentially amorphous or as amixture of amorphous and partially crystalline cellulose, by rapidlyquenching the IL-cellulose mixture with an anti-solvent. When theincubation samples contained initial cellulose weight percent above itssolubility limit in the IL, subsequent anti-solvent treatment provides acellulose mix of amorphous regenerated-cellulose (RC) andpartially-crystalline-cellulose (PCC). The crystallinity index (CrI)obtained from XRD measurement of the IL-treated samples displayed acorresponding increase in CrI when the initial wt % of cellulose wasabove the solubility limit in the IL.

The IL-treated cellulose samples were hydrolyzed to sugars usingcommercial cellulases or cellulases supplemented with β-glucosidase.IL-treated-cellulose exhibited improved hydrolysis kinetics withoptically transparent solutions formed after the first few hours ofreaction, indicating relatively fast hydrolysis kinetics. With optimalIL-treatment conditions and enzyme loadings, initial rates of hydrolysisof IL-treated cellulose were two orders of magnitude higher than thoseobserved with untreated-cellulose. Among IL-treated cellulosepreparations, the initial rates observed with samples containing only RCwere higher than the initial rates for the samples that were mixtures ofRC and PCC. In spite of the observed differences in the initial ratesand CrI, all IL-treated cellulose preparations showed significantlyhigher glucose conversions compared to untreated-cellulose: about 80-85%conversions to glucose were observed for IL-treated cellulose samples in7 hours of hydrolysis whereas conversion for untreated-cellulose wasonly 20%. Thus, it is not necessary to completely eliminate thecrystallinity of cellulose in order to achieve significant enhancementin hydrolysis rates; even with some residual crystallinity therecalcitrance to hydrolysis can be mitigated.

In the proposed technique, dissolution of cellulose in the IL and itssubsequent precipitation with anti-solvent, allows separation of theIL/anti-solvent solution from cellulose by a simple filtration orcentrifugation step. Due to the non-volatility of the IL, anti-solventcan be easily stripped from the IL/anti-solvent solution for recoveryand recycle of both the ionic liquid and anti-solvent. Theseconsiderations point to the promise of the proposed technique in dealingwith the recalcitrance of cellulose to hydrolysis.

Modifications

Specific compositions, methods, or embodiments discussed are intended tobe only illustrative of the invention disclosed by this specification.Variation on these compositions, methods, or embodiments are readilyapparent to a person of skill in the art based upon the teachings ofthis specification and are therefore intended to be included as part ofthe inventions disclosed herein.

The above detailed description of the present invention is given forexplanatory purposes. It will be apparent to those skilled in the artthat numerous changes and modifications can be made without departingfrom the scope of the invention. Accordingly, the whole of the foregoingdescription is to be construed in an illustrative and not a limitativesense, the scope of the invention being defined solely by the appendedclaims.

1. A method for the conversion of cellulose to sugar comprising thesteps of: providing cellulose from a biomass or waste cellulose;incubating the cellulose in an ionic liquid to form a solution;precipitating amorphous cellulose or cellulose of reduced crystallinityby rapidly quenching the admixture with an anti-solvent; and addingcellulase to the amorphous cellulose or cellulose of reducedcrystallinity under conditions which promote the hydrolysis of celluloseto sugars.
 2. A method according to claim 1 wherein the solutionadmixture has a cellulose concentration below the solubility limit.
 3. Amethod according to claim 1 wherein the solution admixture has acellulose concentration above the solubility limit; and furthercomprises an undissolved swollen portion.
 4. A method according to claim1 further comprising the step of separating the precipitated amorphouscellulose or cellulose of reduced crystallinity from the anti-solventprior to adding cellulase.
 5. A method according to claim 1 furthercomprising the step of converting the sugars to ethanol or other fuelsand chemicals.
 6. A method according to claim 1 wherein the ionic liquidis molten at a temperature ranging from −10° C. to 160° C. and comprisescations or anions.
 7. A method according to claim 1 wherein the IL isany ionic liquid that is capable of dissolving cellulose represented bya cation structure that includes imidazolium, pyrroldinium, pyridinium,phosphonium, or ammonium.
 8. A method according to claim 1 wherein theIL is represented by the structure:

wherein each of R1, R2 and, R3 is hydrogen, an alkyl group having 1 to10 carbon atoms or an alkene group having 2 to 10 carbon atoms, whereinthe alkyl group may be substituted with sulfone, sulfoxide, thioether,ether, amide, or amine and wherein A is a halide, acetate,trifluoroacetate, dicyanamide, carboxylate or other anions.
 9. A methodaccording to claim 8 wherein the halide is a chloride, fluoride, bromideor iodide.
 10. A method according to claim 1 wherein the IL is1-n-butyl-3-methylimidazolium chloride.
 11. A method according to claim1 wherein the IL is 1-allyl-3-methyl imidazolium chloride.
 12. A methodaccording to claim 1 wherein the IL is 3-methyl-N-butylpyridiniumchloride.
 12. A method according to claim 1 wherein the IL is3-methyl-N-butylpyridinium chloride.
 13. A method according to claim 1wherein the anti-solvent is water, an alcohol or acetonitrile or anysolvent which dissolves the IL and displaces the dissolved cellulosefrom the IL.
 14. A method according to claim 13 wherein the alcohol isethanol or methanol.
 15. A method according to claim 1 wherein the ILcan be easily recovered from the anti-solvent through distillation,membrane separation, solid phase extraction, and liquid-liquidextraction.
 16. A method according to claim 1 wherein the cellulose iswaste cellulose.
 17. A method according to claim 1 wherein the celluloseis biomass.
 18. A method according to claim 1 wherein the cellulase is amix of component enzymes.
 19. A method according to claim 1 wherein thecellulase is a mix of endo-gluconases, exo-glucanases, andβ-glucosidases. wherein the enzyme mixture can be modified or simplifiedfrom conventional cellulase systems to achieve hydrolysis of IL-treatedcellulose.
 20. A method according to claim 1 wherein the cellulase issupplemented with β-glucosidase.
 21. A method according to claim 1wherein the improved hydrolysis rates allows saccharification with lowenzyme loadings.
 22. A method according to claim 1 wherein enzymes areeasily recovered from cellulose hydrolysates.
 23. A method according toclaim 1 wherein the sugars are simple sugars or soluble oligomers.
 24. Amethod according to claim 1 wherein the sugars are glucose.
 25. A methodaccording to claim 1 wherein the sugars are cellobiose.
 26. A methodaccording to claim 1 wherein the cellulose is native cellulose.
 27. Amethod according to claim 26 wherein the native cellulose is crystallineor partially crystalline.
 28. A method according to claim 1 whereinduring incubation, the solution is subjected to heating with agitationor microwave irradiation with intermittent agitation.